Stock No: 000485
Protocol 25108: Standard PCR Assay - Fas<lpr> MCA
Version 5.2

Notes

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Melt Peaks
Wt = 83°C +/- 1
Mut = 88.5
°C +/- 1


HET = 179 bp and 217 bp
MUT=217 bp
WT=179 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR1678 GTA AAT AAT TGT GCT TCG TCA G Common A
oIMR1679 TAG AAA GGT GCA CGG GTG TG Mutant A
oIMR1680 CAA ATC TAG GCA TTA ACA GTG Wild type A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR1678 0.50 uM
oIMR1679 0.50 uM
oIMR1680 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
000482 B6.MRL-Faslpr/J
002455 MRL.Cg-B2mtm1Unc Faslpr
022350 MRL.Cg-Nos2tm1Lau Faslpr/J
022760 MRL.Cg-Nos3tm1Unc Faslpr/J
000486 MRL/MpJ
006825 MRL/MpJ-Faslpr/2J
000485 MRL/MpJ-Faslpr/J
7 strains use this protocol